This proposal focuses on characterizing the role of microglial toll-like receptor-4 (TLR4) in ischemic preconditioning (IPC). IPC is a robust neuroprotective phenomenon in which a brief period of cerebral ischemia confers transient tolerance to subsequent ischemic challenge. Characterization of the cellular and molecular mechanisms that underlie IPC is an important and active area of investigation in stroke research. Inflammatory responses in the brain are critical in the pathophysiology of stroke. Microglia, the brain's resident tissue macrophages, are central in this process. TLRs mediate powerful immune responses to a variety of pathogen associated molecular patterns including lipopolysaccharide (LPS). TLR4, which is expressed by microglia in the CNS, has been directly implicated in the pathophysiology of stroke. However, the role of microglial TLR4 signaling in IPC is unknown. Here we hypothesize that microglial TLR4 signaling is required for both optimal IPC-mediated neuroprotection and IPC-mediated attenuation of inflammatory responses seen in subsequent prolonged ischemia (stroke). We further postulate that hypoxia-inducible factor-11 (HIF-11), a central regulator of gene expression under low oxygen tension, is critical in mediating the effects of microglial TLR4 signaling on IPC. To test these hypotheses, we will first use a well-established in vivo model of stroke/IPC (mouse middle cerebral artery occlusion/reperfusion paradigm) on TLR4 knockout and control mice. We will assess infarct volume and neurobehavioral outcome. Then we will use ex vivo flow cytometry to characterize the inflammatory infiltrate and microglial phenotype in ischemic cortex following stroke alone, IPC alone or IPC followed by stroke. Second, we will carry out in vitro ischemia experiments on cultured primary microglia from TLR4 knockout and control mice to characterize the role of TLR4 signaling in modulating the microglial response to hypoxia/hypoglycemia. Third, we will use a myeloid-cell targeted, HIF-11 knockdown mouse strain, in addition to the systemic TLR4 knockout strain, to investigate in vivo the role of HIF-11 in mediating the effects of microglial TLR4 signaling on IPC. The results of these studies will be valuable to researchers trying to elucidate the cellular and molecular mechanisms of IPC, neuroinflammation and stroke. It will provide key insights into the pathophysiologic state of microglia in the ischemic penumbra and identify molecular targets for therapeutic intervention in acute stroke. PUBLIC HEALTH RELEVANCE: Stroke is the leading cause of serious long-term disability and the third leading cause of death in the United States. Currently available pharmacologic therapies for acute stroke are few in number and limited by temporal restrictions on their use, modest efficacy and potential for serious side effects. A major goal of this research project is to increase our mechanistic understanding of stroke pathophysiology. By doing so, we hope to identify novel cellular and molecular targets for therapeutic intervention in acute stroke.